Lenalidomide Inhibits Osteoclastogenesis, Survival Factors and Bone-Remodeling Markers in Multiple Myeloma

Lenalidomide inhibits osteoclastogenesis, survival factors and bone-remodeling markers in multiple myeloma

I Breitkreutz1, MS Raab1, S Vallet1, T Hideshima1, N Raje1, C Mitsiades1, D Chauhan1, Y Okawa1, NC Munshi1, PG Richardson1 and KC Anderson1

1Department of Medical Oncology, LeBow Institute for Myeloma Therapeutics, Jerome Lipper Multiple Myeloma Center, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA

Osteolytic bone disease in multiple myeloma (MM) is caused by survival of 28 months and a median progression-free survival of enhanced osteoclast (OCL) activation and inhibition of osteo- 7.7 months.2 In two large-phase III studies comparing lenalido- blast function. Lenalidomide and bortezomib have shown promising response rates in relapsed and newly diagnosed mide/dexamethasone versus dexamethasone in relapsed MM, MM, and bortezomib has recently been reported to inhibit OCLs. extent and frequency of response, as well as progress free and We here investigated the effect of lenalidomide on OCL overall survival, were prolonged in the combined therapy formation and osteoclastogenesis in comparison with bortezo- cohort.3,4 The proteasome inhibitor bortezomib has potent mib. Both drugs decreased aVb3-integrin, tartrate-resistant acid anti-MM activity with impressive clinical responses, prolonging phosphatase-positive cells and bone resorption on dentin time to progression and overall survival in patients with relapsed disks. In addition, both agents decreased receptor activator of 5,6 nuclear factor-jB ligand (RANKL) secretion of bone marrow or refractory MM. In spite of these novel agents, osteolytic stromal cells (BMSCs) derived from MM patients. We identified bone disease remains a major source of morbidity, occurring in PU.1 and pERK as major targets of lenalidomide, and nuclear 70–80% of MM patients and associated with severe bone pain, factor of activated T cells of bortezomib, resulting in inhibition pathologic fractures, paralysis through nerve compression, of osteoclastogenesis. Furthermore, downregulation of cathe- hypercalcemia and death.7 Bisphosphonates inhibit osteoclast psin K, essential for resorption of the bone collagen matrix, was (OCL) activity and have been successfully and widely used for observed. We demonstrated a significant decrease of growth 8 and survival factors including macrophage inflammatory the treatment of MM bone disease; however, complications protein-a, B-cell activating factor and a proliferation-inducing induced by bisphosphonates can occur including osteonecrosis ligand. Importantly, in serum from MM patients treated with of the jaw, and their use is not recommended over a long period lenalidomide, the essential bone-remodeling factor RANKL, as of time. well as the RANKL/OPG ratio, were significantly reduced, Osteolytic lesions are primarily due to a dysregulation of the whereas osteoprotegerin (OPG) was increased. We conclude normal bone-remodeling process, with a decrease of osteoblast that both agents specifically target key factors in osteoclastogenesis, and could directly affect the MM-OCL-BMSCs activa- (OBL) function accompanied by increased activation of OCLs. tion loop in osteolytic bone disease. In MM, this destructive bone process is enhanced by interaction Leukemia (2008) 22, 1925–1932; doi:10.1038/leu.2008.174; of MM cells with OCLs in the bone marrow (BM) microenviron- published online 3 July 2008 ment. Specifically, adhesion of MM cells to bone marrow Keywords: multiple myeloma; osteoclastogenesis; bone disease; stromal cells (BMSCs) induces secretion of osteolytic factors lenalidomide; bortezomib such as interleukin-6 (IL-6) and receptor activator of NF-kB ligand (RANKL), a tumor necrosis factor (TNF) family cytokine. IL-3 production stimulates osteoclastogenesis directly and enhances the effect of RANKL and of macrophage inflammatory Introduction protein-a (MIP-1a), an important growth and survival factor for MM cells and OCL.9 In addition, MM cells in the BM produce Multiple myeloma (MM) is a currently incurable malignant Dickkopf homologue 1, thereby inhibiting OBL activation by plasma cell disorder affecting approximately 15 000 new blocking Wnt signaling.10 patients in the United States annually. Novel drugs targeting Clinical observations revealed that bortezomib may trigger MM and its microenvironment have shown promising clinical OBL activation, evidenced by increased alkaline phosphatase in results.1 Thalidomide and its more potent immunodmodulatory the serum of MM patients responding to bortezomib treat- analog (IMiDs) lenalidomide have been successfully used in ment.11 Moreover, treatment with bortezomib in patients with MM treatment. Specifically, the results of a multicenter, open- relapsed MM significantly decreased RANKL and C-terminal labeled, randomized phase 2 study evaluating two dose cross-linking telopeptide of collagen type I (CTX).12 Recently, regimens of lenalidomide for relapsed, refractory myeloma von Metzler et al.13 showed that bortezomib inhibits osteoclas- show an overall response rate of lenalidomide alone of 25%. It togenesis by downregulation of p38 mitogen-activated protein was well tolerated at 30 mg once daily, with a median overall kinase (MAPK) and AP1. To date, however, the effect of lenalidomide on OCLs remains unknown. Furthermore, it is Correspondence: Dr KC Anderson or Dr I Breitkreutz, Department of not clear whether there is an inhibitory effect of lenalidomide or Medical Oncology, LeBow Institute for Myeloma Therapeutics, Jerome bortezomib on growth and survival factors that trigger Lipper Multiple Myeloma Center, Dana-Farber Cancer Institute, osteoclastogenesis and OCL activation. In this study, we Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA. investigated whether lenalidomide, similar to bortezomib, E-mails: [email protected] or Iris_Breitkreutz@ dfci.harvard.edu affects activation and function of OCLs in the BM micro- Received 28 February 2008; revised 27 May 2008; accepted 30 May environment, osteoclastogenesis and related growths factors, as 2008; published online 3 July 2008 well as markers of bone turnover in MM. Lenalidomide, bortezomib inhibit osteoclasts in MM I Breitkreutz et al 1926 Materials and methods ELISA Cytokine secretion in supernatants from OCL cultures was Osteoclast formation assay measured using enzyme-linked immunosorbent assay (ELISA). OCLs were generated in vitro using peripheral blood mono- Specifically, OCLs were cultured in 96-well plates for 2 weeks; nuclear cells (PBMCs) from MM patients. Written informed supernatants were harvested, and MIP-1a, IL-6, B-cell activa- consent was obtained according to the Declaration of Helsinki. ting factor (BAFF) and a proliferation-inducing ligand (APRIL) For OCL formation assays, PBMCs were separated by secretion were measured using DuoSet ELISA development kits Ficoll-Paque gradient, and nonadherent cells were cultured in (R&D Systems Inc.) and Bender MedSystems (Burlingame, CA, 6- or 96-well plates (0.5 Â 06 cells per cm2), as previously USA), in accordance with manufacturer’s instructions. To described.14 OCLs were generated by culturing cells for 14–21 measure RANKL secretion, BMSCs were cultured in RPMI/ days in a-minimal essential medium containing 10% 20% FBS in the presence of lenalidomide or bortezomib. After fetal bovine serum (FBS), 1% penicillin-streptomycin (Media- 72 h, supernatant was harvested and subjected to RANKL ELISA tech Inc., Herndon, VA, USA), as well as 25 ng/ml of (Bender MedSystems Burlingame). macrophage colony-stimulating factor (M-CSF) (R&D Systems, Minneapolis, MN, USA) and RANKL (50 ng/ml) (PeproTech, Rocky Hill, NJ, USA). Western blot analysis PBMCs were cultured with RANKL (50 ng/ml) and M-CSF (25 ng/ ml) in the presence or absence of lenalidomide or bortezomib. Bone marrow stromal cell cultures PBMCs were seeded in six-well plates. Cells were harvested at BMSCs derived from MM patients were cultured in RPMI and specific time points with cell dissociation buffer (Invitrogen) and 20% FBS after separation of mononuclear cells via Ficoll-Paque M 4 lyzed in lysis buffer (50 m HEPES (N-2-hydroxyethylpipera- gradient. BMSCs were cultured in 96-well plates (0.5 Â 10 cells 0 M 2 zine-N 2-ethanesulfonic acid), pH 7.4, 150 m NaCl, 1% per cm ). Medium was changed twice weekly. Lenalidomide NP-40, 30 mM sodium pyrophosphate, 5 mM ethylenediamine- and bortezomib were diluted in culture medium and added to tetraacetic acid, 2 mM Na3VO4, 5 mM NaF, 1 mM phenylmethyl 1 BMSCs for 72 h. Supernatant was collected and stored at –80 C. sulfonyl fluoride, 5 mg/ml leupeptin and 5 mg/ml aprotinin). Total protein lysates were then subjected to sodium dodecyl sulfate– polyacrylamide gel electrophoresis, transferred to nitrocellulose Osteoclast differentiation and bone resorption assay membrane and immunoblotted with antibodies against pERK, After 2 weeks of incubation, OCLs in control and treated groups nuclear factor of activated T cells (NFATe1), c-fos (Santa Cruz were fixed with citrate-acetone solution and stained for tartrate- Biotechnology, Santa Cruz, CA, USA), as well as PU.1 and resistant acid phosphatase (TRAP) using an acid phosphatase cathepsin K (Cell Signaling Technology, Beverly, MA, USA). leukocyte staining kit (Sigma Chemical, Saint Louis, MO, USA). Antigen–antibody complexes were detected by enhanced TRAP-positive multinucleated OCLs containing three or more chemiluminescence (Amersham, Arlington Heights, IL, USA). nuclei per cell were enumerated using an inverted microscope. The membrane was stripped and reprobed with anti-actin or Images were obtained using a Leica DM IL microscope (Leica ERK2 (Cell Signaling Technology) antibody to ensure equal Microsystems, Wetzlar, Germany) and were acquired through protein loading. IM50 software (Leica Microsystems Imaging Solutions, Cambridge, UK). For bone resorption assays, cells were cultured on dentine slices with RANKL and M-CSF for 3 weeks, in the absence or presence of lenalidomide or bortezomib, and Patient characteristics resorbtion lacunae were enumerated using an inverted micro- Serum from 20 MM patients was obtained before therapy as well scope. PBMCs were cultured (0.5 Â 106 cells per well) on as on day 28 of the second cycle and subjected to osteopro- dentin slices (Immunodiagnostic Systems Ltd., Boldon, England) tegerin (OPG) and RANKL ELISA (Bender MedSystems Burlin- in 96-well plates, as per the manufacturer’s guidelines. After game). Patients enrolled in the study had Durie stage II and III incubation time, cells were scraped off gently with 0.1% triton. MM, were at least 18-year old, had relapsed or refractory Bone slices were washed in distilled water and stained with 1% disease after a median of 4 prior chemotherapies, and had given toluidine solution. After staining resorption areas with toluidine written informed consent in accordance to the Declaration of blue, images were obtained using a Leica DM IL microscope Helsinki. All patients had radiologically confirmed, extensive and IM50 software. Bone resorption was measured by osteolytic bone disease (44 osteolytic lesions), and had been enumerating resorption pits. treated with bisphosphonates monthly since their disease diagnosis. In total, 55% of the patients had IgG, 25% IgA and 20% Bence Jones proteinuria. Overall response rate was 45% (complete remission, partial remission and minimal response), Flow cytometry with 55% stable disease (Supplementary data, Table 1). For analysis of cell surface marker expression after 2 weeks of differentiation and incubation in the absence or presence of lenalidomide or bortezomib, OCLs were harvested with cell dissociation buffer (Invitrogen, Carlsbad, CA, USA) and Statistical analysis suspended in Dulbecco’s phosphate-buffered saline. For OCL Each experiment was repeated at least three times. All detection, cells were stained with aVb3-integrin (fluorescein- quantitative data are presented as median plus range. Wilcoxon conjugated mouse immunoglobulin G1 (IgG1), CD51/CD61; rank-sum test was used for comparisons in all in vitro Chemicon International, Temecula, CA, USA). Following 1-hour experiments and of cytokine serum levels at baseline versus incubation at 4 1C with the primary antibody, cells were washed after two cycles of lenalidomide monotherapy. All P-values and analyzed using the RXP cytomics software on an Epics flow were two-sided, and Po0.05 was considered statistically cytometer (Coulter Immunology, Hialeah, FL, USA). significant.

Leukemia Lenalidomide, bortezomib inhibit osteoclasts in MM I Breitkreutz et al 1927 Results and discussion previous reports,13 bortezomib inhibited formation of TRAP-positive, multinucleated cells (bortezomib at 0 nM: Lenalidomide and bortezomib have been shown to interfere median 70.5%; range 50.0–84.0%; at 2 nM: median 26.0%; with MM tumor growth and survival in the BM microenviron- range 11.0–39.0%; Po0.01; at 5 nM: median 17.0%; range ment and to be effective in the treatment of relapsed and 13.0–34.0%; Po0.001) (Figure 1a). Furthermore, formation of refractory MM.15,16 We here report the influence of lenalido- characteristic resorption lacunae was reduced by both lenali- mide on osteoclastogenesis in vitro and in vivo, secretion of key domide (lenalidomide at 0 mM: median 100%; at 2 mM: median factors from BMSCs important for OCL survival as well as OCL 38%; range 15–50%; at 10 mM: median 20%; range 0–35%; formation and activation in MM, and compare these effects with Po0.02) and bortezomib (bortezomib at 0 nM: median 100%; at bortezomib. 2nM: median 3%; range 0–5%; at 5 nM: median 0%; range 0–0% Po0.0001) (Figure 1b). Multiple factors are involved in the activation of OCLs that Inhibition of OCL formation, bone resorption, eventually lead to bone resorption. Cathepsin K is the major expression of aVb3-integrin and cathepsin K cysteine protease expressed in OCLs and is important for matrix To rule out severe cytotoxicity in OCL cultures, we first degradation during bone resorption. It is processed intracellu- investigated susceptibility of OCL cultures to short-term larly, presumably in lysosomal compartments, prior to the exposure (72 h) of lenalidomide and bortezomib at different release of active enzyme into the resorption lacunae.18 aVb3- time points and concentrations. Overall, both agents showed integrin is an important marker of OCL differentiation and a only minor cytotoxicity at different time points of OCL member of a superfamily of adhesion molecules that mediates development (data not shown). interactions between cells and the extracellular matrix, activates Next we investigated the effect of lenalidomide and bortezo- OCLs and is associated with OCL bone resorption.19 We here mib on OCL formation and their ability to resorb bone. During investigated whether lenalidomide altered the expression of osteoclastogenesis, TRAP-negative mononuclear cell progress to aVb3-integrin and cathepsin K in OCLs and found a dose- pro-OCL and then pre-OCL (small round mononucleated dependent decrease of aVb3-integrin-positive cells (Figure 1c) TRAP-positive cells), ultimately fusing to form mature OCL (lenalidomide at 0 mM: median 69.3%; range 28.9–89.0%; at (multinucleated TRAP-positive cells) capable of extensive 2 mM: median 50.4%; range 21.5–64.2%; at 10 mM: median lacunar resorption.17 In our study, lenalidomide significantly 39.2%; range 33.6–47.5%); bortezomib decreased these cells decreased TRAP-positive and multinucleated cells in a dose- more potently (bortezomib at 0 nM: median 69.3%; range dependent manner (lenalidomide at 0 mM: median 70.5%; range 28.9–89.0%; at 2 nM: median 35.0%; range 11.0–79.0%; at 50.0–84.0%; at 2 mM: median 47.0%; range 31.0–77.0%; at 5nM: median 11.5%; range 5.5–18.8%; Po0.05). Furthermore, 10 mM: median 32.5%; range 14.0–44.0%; Po0.05). As in western blot analysis showed a dose-dependent downregulation

100 p<0.05 100 p<0.01 p<0.001 100 100

75 75 75 75 p<0.05

50 50 50 50 cells cells

25 25 25 3 integrin-positive 25 3 integrin-positive β β V V α %TRAP-positive cells %TRAP-positive cells 0 0 α 0 0 %

0210 025 % 0210 025 lenalidomide [µM] bortezomib [nM] lenalidomide [µM] bortezomib [nM]

21d 10x 10x 10x

lenalidomide bortezomib µM nM Resorption lacunae 0µMlenalidomide 2µM lenalidomide 10µM

10x 10x 10x 021025 CathepsinK

Actin

Resorption lacunae 0nMbortezomib 2nM bortezomib 5nM

Figure 1 Lenalidomide and bortezomib inhibit osteoclast (OCL) formation and bone resorption, as well as the expression of aVb3-integrin and cathepsin K in OCL cultures. (a) Lenalidomide and bortezomib in a dose-dependent fashion induced a statistically signiﬁcant decrease in multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells. Results are expressed as percentage of TRAP-positive cells per well. Data shown represent median and range. (b) After incubation of OCLs on dentin disks for 3 weeks and staining with toluidine blue, resorption lacunae were measured. Data shown are representative of at least three independent experiments. A decrease of resorptive tracks and pits was observed in the presence of lenalidomide, with complete abrogation in the presence of bortezomib. (c) Expression of aVb3-integrin was measured using ﬂow cytometry. Lenalidomide and bortezomib triggered in a dose-dependent decrease in aVb3-integrin expressing cells. Data shown represent median and range. Each experiment was repeated at least three times from different donors. (d) After stimulation of 21 days, both lenalidomide and bortezomib downregulated expression of cathepsin K in a dose-dependent manner, evaluated by western blot analysis.

Leukemia Lenalidomide, bortezomib inhibit osteoclasts in MM I Breitkreutz et al 1928 of cathepsin K induced by both novel agents (Figure 1d). Our M-CSF.22 M-CSF triggers activation of phosphatidylinositol-3 results therefore suggest that lenalidomide, similar to bortezo- (PI-3) kinase/AKT that mediates OCL survival during early and mib, interferes with OCL differentiation, cell adhesion, cell late osteoclastogenesis. M-CSF further stimulates MAPK, ERK fusion and bone resorption during osteoclastogenesis. It is and Fos/Jun; RANKL activates TNF receptor-associated factor 6 noteworthy that in our bone formation assays, bortezomib (TRAF6) and the AP1/c-fos complex. The AP1/c-fos complex is a seemed to have a stronger inhibitory effect than lenalidomide. key regulator of OCL differentiation. Mice lacking c-fos develop Furthermore, compared with MM cell lines, even lower doses of osteopetrosis due to defective OCL differentiation, whereas the bortezomib could be used, whereas higher ones of lenalidomide number of macrophages increases.23,24 AP1/c-fos in turn were required. activates Jun N-terminal kinase 1, nuclear factor (NF)-kB and its downstream target NFATc1, a major transcription factor modulating osteoclastogenesis.25 Downregulation of key transcription factors during Pomalidomide (CC-4047, actimid), another IMiD, has been osteoclastogenesis shown to inhibit osteoclastogenesis by downregulating PU.1;14 The signal transduction pathways modulating osteoclastogenesis Anderson et al. showed that pomalidomide, but not thalido- have been extensively studied: PU.1 plays a critical role in the mide, decreased the expression of PU.1. However, no inhibition early determination phase of osteoclastogenesis, whereas of c-fos could be found after either pomalidomide or thalido- activation of MAPK, and extracellular signal-regulated kinase mide treatment. von Metzler et al.13 reported an inhibitory effect (ERK), mediates OCL survival and differentiation.20 PU.1 of bortezomib on human osteoclastogenesis, associated with regulates the differentiation of myeloid cells to OCL precursors; p38 MAPK and AP1 inhibition. They found that bortezomib conversely, PU.1-deﬁcient mice develop osteopetrosis.21 targeted OCLs especially in the range between 1 and 100 nM, RANKL induces OCL differentiation in the presence of with decreasing effects of bortezomib on more differentiated

3h 3h

0210lenalidomide µM 025bortezomib nM pERK pERK ERK2 ERK2

4d 4d

0210lenalidomide µM 025bortezomib nM PU.1 PU.1 Actin Actin

7d 7d

0210lenalidomide µM 025bortezomib nM NFATc1 NFATc1 Actin Actin

lenalidomide bortezomib µM nM

021025 c-fos Actin

Figure 2 Effect of lenalidomide and bortezomib on transcription factors during osteoclastogenesis. (a) Lenalidomide (100% at 0 mM, 60.2% at 2 mM and 48.8% at 10 mM), but not bortezomib, resulted in a dose-dependent inhibition of extracellular signal-regulated kinase (ERK) phosphorylation in peripheral blood mononuclear cells (PBMCs) incubated with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL) for 3 h. (b) Lenalidomide, but not bortezomib, resulted in a decrease of PU.1 after incubation of PBMCs for 4 days in the presence of RANKL and M-CSF. Conversely, bortezomib, but not lenalidomide, downregulated NFATc1. C-fos was not downregulated by either agent at 5 days.

Leukemia Lenalidomide, bortezomib inhibit osteoclasts in MM I Breitkreutz et al 1929 stages of OCLs. The IC50 of bortezomib found in their also upregulated the anti-apoptotic factor Bcl-2 in MM cells. experiments was much lower compared with the IC50 of MM These protective effects were mediated by activation of NF-kB, cell lines. We here showed that lenalidomide speciﬁcally PI3K/AKT and MAPK signaling. Importantly, OCL produced inhibits phosphorylation of ERK, as well as expression of PU.1 (Figures 1a, b), indicating that lenalidomide targets osteoclastogenesis at an early stage of differentiation. In contrast, 250 p<0.05 250 p<0.05 bortezomib downregulated NFATc1 (Figure 2b), known to be 200 200 activated at a later stage of differentiation. This is consistent with 150 150 -secretion

100 pg/ml

-secretion 100 the ﬁndings of von Metzler et al., as NFATc1 is a downstream α pg/ml α target of the AP1 complex. Neither drugs inhibited c-fos. 50 50 MIP1- Therefore, lenalidomide and bortezomib speciﬁcally inhibited MIP1- 0 0 0210 025 key factors at different stages of OCL development. lenalidomide [M] bortezomib [nM]

250 250 p<0.05 p<0.05 p<0.05 Lenalidomide and bortezomib inhibit MIP-1a, 200 200 BAFF- and APRIL secretion by OCLs as well as 150 150

RANKL secretion by bone marrow stromal cells 100 pg/ml 100 pg/ml from MM patients 50 50 BAFF-secretion MIP-1a, a key promoter in osteoclastogenesis, is produced mainly BAFF-secretion 0 0 by MM cells and OCLs, and triggers OCL differentiation and 0210 025 lenalidomide [M] bortezomib [nM] survival.26 It acts directly on human OCL progenitors and is 27 a involved at later stages of OCL differentiation. Levels of MIP-1 10.0 10.0 in freshly isolated BM from patients with active MM are higher 7.5 7.5 p<0.01 (range 75–7784 pg/ml) than with stable disease (range 75– 190.3 pg/ml),28 and serum levels of MIP-1a correlated with 5.0 5.0 ng/ml osteolytic lesion in MM patients.29 In addition, MIP-1a stimulates ng/ml 2.5 2.5 30 0.0 APRIL-secretion 0.0 proliferation, migration and survival of MM cells. Furthermore, APRIL-secretion MIP-1a enhances adhesive interactions between MM cells and 0210 025 lenalidomide [M] bortezomib [nM] BMSCs, leading to an increased expression of RANKL and IL-6, thereby increasing bone destruction and tumor burden. Here we 20 20 examined the effect of lenalidomide and bortezomib on MIP-1a 15 15 secretion in OCL cultures, and found a dose-dependent decrease 10 10 U/L U/L RANKL a RANKL in MIP-1 secretion triggered by lenalidomide and bortezomib 5 5 (lenalidomide at 0 mM: median 171.4 pg/ml; range 159.3– 0 0 219.6 pg/ml; at 2 mM: median 91.6 pg/ml; range 73.4–100.0 pg/ 0210 025 ml; at 10 mM: median 83.8 pg/ml; range 76.6–86.5 pg/ml; lenalidomide [M] bortezomib [nM] Po0.05) (bortezomib at 0 nM: median 171.4 pg/ml; range 159.3–219.6 pg/ml; at 2 nM: median 115.1 pg/ml; range 72.6– Figure 3 Lenalidomide and bortezomib inhibit secretion of macrophage inﬂammatory protein-a (MIP-1a), B-cell activating factor (BAFF) 141.2 pg/ml; at 5 nM: median 87.3 pg/ml; range 60.6–103.4 pg/ and a proliferation-inducing ligand (APRIL) by osteoclasts (OCLs), as ml; Po0.05) (Figure 3a) These results indicate that both well as inhibit receptor activator of NF-kB ligand (RANKL) secretion lenalidomide and bortezomib decrease MIP-1a, one of the most by bone marrow stromal cells (BMSCs) cells from multiple myeloma important growth and survival factors of OCLs. (MM) patients. Lenalidomide and bortezomib inhibited secretion of (a) BAFF31 and a proliferation-inducing ligandAPRIL32 are MIP-1a,(b) BAFF and (c) APRIL in a dose-dependent manner. Data members of the TNF family (TNF-a) and promote MM cell shown represent median and range. All experiments were performed independently at least three times from different donors. (d) BMSCs growth and survival in the BM microenvironment, as well as 33,34 35 derived from MM patients were cultured in RPMI and 20% fetal growth of other B-cell malignancies. Moreaux et al. bovine serum (FBS). Both lenalidomide and bortezomib induced a reported that BAFF and APRIL protect MM cells from apoptosis dose-dependent inhibition of RANKL secretion in BMSCs. Data shown initiated by IL-6 deprivation and dexamethasone treatment, and represent mean and s.d.

p<0.05 p<0.05 p<0.05 40 17 5

15 4 30 13 3 20 11 2

10 OPG (pg/ml)

RANKL (pg/ml) 9 1 RANKL/OPG ratio 0 7 0 baseline at 2 months baseline at 2 months baseline at 2 months

Figure 4 Lenalidomide inhibits bone-remodeling markers from patients with relapsed or refractory multiple myeloma (MM). Serum from patients with refractory or relapsed MM was subjected to receptor activator of NF-kB ligand (RANKL) and osteoprotegerin (OPG) enzyme-linked immunosorbent assay (ELISA) before (baseline) and after 2 months of lenalidomide treatment. Lenalidomide treatment significantly decreased RANKL after 2 months (Po0.05). Furthermore, RANKL/OPG ratio was also significantly reduced (Po0.05), whereas OPG secretion was significantly increased (Po0.05).

Leukemia Lenalidomide, bortezomib inhibit osteoclasts in MM I Breitkreutz et al 1930 threefold more BAFF and eightfold more APRIL than BMSCs, we found only minor cytotoxicity versus significant inhibition of thereby promoting MM cell growth and survival.36 Production differention into multinucleated and functional OCLs (Figure 1). of BAFF and APRIL activates both OCLs and MM cells.37 Moreover, cell numbers at time of supernatant harvest were Importantly, Yaccoby et al.38 recently reported that BAFF and similar in all treatment groups, indicating a minor contribution APRIL were highly upregulated in MM cells co-cultured with of cytotoxicity to decrease cytokine levels. OCLs. We here measured levels of BAFF and APRIL secreted in RANKL is the key mediator of osteoclastogenesis and plays a supernatants of RANKL and M-CSF-stimulated OCL cultures and crucial role in bone destruction in malignant bone disease. MM found that both lenalidomide and bortezomib significantly cells also express RANKL,39 and the level of RANKL expression 40 reduced BAFF (lenalidomide at 0 mM: median 127.6 pg/ml; correlates with the status of osteolytic bone disease in MM. range 32.0–420.0 pg/ml; at 2 mM: median 64.9 pg/ml; range RANKL is produced mainly by BMSCs after adhesion of MM 32.1–288.2 pg/ml; at 10 mM: median 60.1 pg/ml; range 27.6– cells; conversely, blockade of RANKL suppressed OCL matura- 119.7 pg/ml; Po0.05) (bortezomib at 0 nM: median 127.6 pg/ml; tion, bone resorption and tumor development. We here range 32.0–420.0 pg/ml; at 2 nM: median 53.4 pg/ml; range investigated the potential of lenalidomide and bortezomib to 26.3–214.4 pg/ml; at 5 nM: median 53.3 pg/ml; range interfere with RANKL secretion by BMSCs derived from MM 24.9–103.8 pg/ml; Po0.05, respectively) (Figure 3b) and APRIL patients. Lenalidomide and bortezomib inhibited RANKL secre- (lenalidomide at 0 mM: median 6.2 ng/ml; range 3.9–7.2 ng/ml; tion in a dose-dependent manner (lenalidomide at 0 mM: median at 2 mM: median 4.9 ng/ml; range 3.6–5.6 ng/ml; at 10 mM: 13.9 U/l, at 2 mM: 8.5 U/l, at 10 mM: 7.6 U/l; bortezomib at 0 nM: median 4.2 ng/ml; range 3.6–5.2 ng/ml) (bortezomib at 0 nM: median 15.0 U/l, at 5 nM: 9.1 U/l, at 5 nM: 2.9 U/l) (Figure 3d), median 6.2 ng/ml; range 3.9–7.2 ng/ml; at 2 nM: median 4.1 ng/ thereby contributing to the inhibition of osteoclastogenesis. ml; range 3.5–5.0 ng/ml; at 5 nM: median 2.1 ng/ml; range 0.6–3.7 ng/ml; Po0.01) (Figure 3c) secretion. As BAFF and APRIL trigger OCL and MM cell proliferation and survival, these Lenalidomide significantly inhibits bone-remodeling results suggest that both agents decrease important promoters of markers in patient’s serum MM cells and OCLs in the BM microenvironment. It is RANKL and OPG are important indicators of bone remodeling noteworthy that the inhibitory effect on BAFF, APRIL and MIP- in vivo. OPG is the soluble decoy receptor for RANKL41 and 1a secretion may in part be caused by cytotoxicity. However, negatively regulates osteoclastogenesis by inhibiting RANKL

M-CSF c-Fms RANK RANKL

MEK1 αVβ3 TRAF6 Hematopoietic PU.1 stem cell ERK

AP1/c-fos NFATc1 Commitment TRAP TRAP/Cathepsin K Differentiation Fusion/Function/Resorption

Macrophages Osteoclast precursor Osteoclast MIP-1α, BAFF, APRIL MIP-1α RANKL IL-3

lenalidomide

bortezomib

Bone marrow stromal cells Myeloma cell

Figure 5 Schematic representation of the effect of lenalidomide and bortezomib on osteoclastogenesis in multiple myeloma (MM). A schematic representation of speciﬁc targets of lenalidomide and bortezomib during osteoclastogenesis is shown. In the early stage of osteoclastogenesis, PU.1 induces commitment from hematopoetic cells to macrophages. Macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL) induce a cascade of transcription factors leading to activation, differentiation and function of osteoclasts (OCLs). OCLs secrete survival factors for MM cells; bone marrow stromal cells (BMSCs) and MM cells secrete growth factors for MM cells and OCLs. Lenalidomide and bortezomib inhibit key factors of osteoclastogenesis, abrogate OCL differentiation and function, and inhibit secretion of MM survival factors from OCLs and BMSCs.

Leukemia Lenalidomide, bortezomib inhibit osteoclasts in MM I Breitkreutz et al 1931 and stimulating OBLs. In MM, the ratio of RANKL/OPG is 2 Richardson PG, Blood E, Mitsiades CS, Jagannath S, Zeldenrust SR, increased due to both enhanced RANKL production and Alsina M et al. A randomized phase 2 study of lenalidomide decreased OPG production triggered by adhesion of MM cells therapy for patients with relapsed or relapsed and refractory to BMSCs.42 Serum levels of soluble RANKL/OPG in MM multiple myeloma. Blood 2006; 108: 3458–3464. 3 Dimopoulos M, Spencer A, Attal M, Prince HM, Harousseau JL, patients correlate with the extent of bone disease and survival.43 44 Dmoszynska A et al. Lenalidomide plus dexamethasone for Terpos et al. investigated the influence of thalidomide and relapsed or refractory multiple myeloma. N Engl J Med 2007; dexamethasone on bone disease in 35 patients with relapsed or 357: 2123–2132. refractory MM, and found a normalization of bone-remodeling 4 Weber DM, Chen C, Niesvizky R, Wang M, Belch A, Stadtmauer markers. Specifically, they measured RANKL, OPG and markers EA et al. Lenalidomide plus dexamethasone for relapsed multiple of bone resorption and function, and found that prior to Thal/ myeloma in North America. N Engl J Med 2007; 357: 2133–2142. Dex treatment, MM patients had an increased RANKL/OPG 5 Jagannath S, Barlogie B, Berenson J, Siegel D, Irwin D, Richardson PG et al. A phase 2 study of two doses of bortezomib in relapsed or ratio, whereas treatment with Thal/Dex resulted in normal- refractory myeloma. Br J Haematol 2004; 127: 165–172. ization of this ratio. We here investigated the effect of 6 Richardson PG, Sonneveld P, Schuster MW, Irwin D, Stadtmauer EA, lenalidomide on osteolytic bone disease from patients with Facon T et al. Bortezomib or high-dose dexamethasone for relapsed relapsed or refractory MM treated in a randomized trial multiple myeloma. NEnglJMed2005; 352: 2487–2498. comparing 30 mg daily versus 15 mg twice daily lenalidomide. 7 Callander NS, Roodman GD. Myeloma bone disease. Semin The median OPG at baseline was significantly lower (median Hematol 2001; 38: 276–285. 8.8 pg/ml, range 7.7–12.6 pg/ml) than after treatment (median 8 Berenson JR, Lichtenstein A, Porter L, Dimopoulos MA, Bordoni R, George S et al., Myeloma Aredia Study Group. Efficacy of 10.4 pg/ml, range 8.5–15 pg/ml) (P 0.05) (Figure 4). Lenalido- o pamidronate in reducing skeletal events in patients with advanced mide also significantly inhibited the secretion of RANKL in multiple myeloma. N Engl J Med 1996; 334: 488–493. patient’s serum (baseline: median 17 pg/ml, range 9.0–36 pg/ml; 9 Lee JW, Chung HY, Ehrlich LA, Jelinek DF, Callander NS, 2 months after therapy: median 4.2 pg/ml, range 2.5–36 pg/ml) Roodman GD et al. IL-3 expression by myeloma cells increases (Po0.05). Finally, the ratio of RANKL/OPG was significantly both osteoclast formation and growth of myeloma cells. Blood higher before than after therapy (baseline: median 2.2 pg/ml, 2004; 103: 2308–2315. range 1.1–3.0 pg/ml; 2 months after therapy: median 0.5 pg/ml, 10 Tian E, Zhan F, Walker R, Rasmussen E, Ma Y, Barlogie B et al. The role of the Wnt-signaling antagonist DKK1 in the development of range 0.3–2.6 pg/ml) (P 0.05) (Figure 4). Therefore, lenalido- o osteolytic lesions in multiple myeloma. N Engl J Med 2003; 349: mide specifically reduces serum markers of osteolytic bone 2483–2494. disease in MM patients. It is noteworthy that all patients received 11 Zangari M, Esseltine D, Lee CK, Barlogie B, Elice F, Burns MJ et al. bisphosphonates from time of disease diagnosis, including after Response to bortezomib is associated to osteoblastic activation in relapse, so our results are mainly due to relapse therapy with patients with multiple myeloma. Br J Haematol 2005; 131: 71–73. lenalidomide, rather than bisphosphonate treatment. 12 Terpos E, Heath DJ, Rahemtulla A, Zervas K, Chantry A, In conclusion, it has not been clear whether new drugs in MM Anagnostopoulos A et al. Bortezomib reduces serum dickkopf-1 and receptor activator of nuclear factor-kappaB ligand concentra- directly target mediators of bone disease or whether these effects tions and normalises indices of bone remodelling in patients with are due to a decrease in MM tumor burden. Importantly, we relapsed multiple myeloma. Br J Haematol 2006; 135: 688–692. demonstrated that lenalidomide inhibits OCL formation and 13 von Metzler I, Krebbel H, Hecht M, Manz RA, Fleissner C, Mieth activation through inhibition of key factors during osteoclasto- M et al. Bortezomib inhibits human osteoclastogenesis. Leukemia genesis, including PU.1, and pERK. We further showed that 2007; 21: 2025–2234. lenalidomide and bortezomib block OCL-derived secretion of 14 Anderson G, Gries M, Kurihara N, Honjo T, Anderson J, growth and survival factors (MIP-1a, BAFF, APRIL) and RANKL Donnenberg V et al. Thalidomide derivative CC-4047 inhibits secretion from BMSCs, indicating that both agents not only osteoclast formation by down-regulation of PU.1. Blood 2006; 107: 3098–3105. reduce MM burden, but also prevent osteoclastogenesis directly. 15 Hideshima T, Chauhan D, Podar K, Schlossman RL, Richardson P, Of clinical relevance, we demonstrated that lenalidomide Anderson KC. Novel therapies targeting the myeloma cell and treatment decreased serum bone-remodeling markers in patients its bone marrow microenvironment. Semin Oncol 2001; 28: with refractory and relapsed MM. Potent anti-MM agents in 607–612. combination with bisphosphonates reduce OCL activity through 16 Richardson PG, Sonneveld P, Schuster MW, Irwin D, reduction of tumor burden, and future trials will determine Stadtmauer EA, Facon T et al. Bortezomib or high-dose dexa- whether bortezomib and lenalidomide, used either alone or methasone for relapsed multiple myeloma. N Engl J Med 2005; 352: 2487–2498. combined with bisphosphonates, can further reduce OCL 17 Grigoriadis AE, Wang ZQ, Cecchini MG, Hofstetter W, Felix R, activity and more effectively prevent osteolytic lesions in MM. Fleisch HA et al. c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling. Science 1994; 266: Acknowledgements 443–448. 18 Rieman DJ, McClung HA, Dodds RA, Hwang SM, Holmes MW, We thank the patients, nursing staff as well as the clinical research James IE et al. Biosynthesis and processing of cathepsin K in coordinators of the Jerome Lipper Multiple Myeloma Center/ cultured human osteoclasts. Bone 2001; 28: 282–289. Dana-Farber Cancer Institute, for their help in providing serum 19 Faccio R, Grano M, Colucci S, Villa A, Giannelli G, Quaranta V specimens for this study. This work was supported by the Multiple et al. Localization and possible role of two different alpha v beta 3 Myeloma Research Foundation, LeBow Fund to Cure Myeloma integrin conformations in resting and resorbing osteoclasts. J Cell (KCA); National Foundation of Cancer Research and NIH grants Sci 2002; 115 (Part 14): 2919–2929. 20 Lee SE, Woo KM, Kim SY, Kim HM, Kwack K, Lee ZH et al. The CA50947, CA78373 and CA10070. phosphatidylinositol 3-kinase, p38, and extracellular signal- regulated kinase pathways are involved in osteoclast differentiation. Bone 2002; 30: 71–77. References 21 Tondravi MM, McKercher SR, Anderson K, Erdmann JM, Quiroz M, Maki R et al. Osteopetrosis in mice lacking haematopoietic 1 Hideshima T, Anderson KC. Molecular mechanisms of novel transcription factor PU.1. Nature 1997; 386: 81–84. therapeutic approaches for multiple myeloma. Nat Rev Cancer 22 Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and 2002; 2: 927–937. activation. Nature 2003; 423: 337–342.

Leukemia Lenalidomide, bortezomib inhibit osteoclasts in MM I Breitkreutz et al 1932 23 Grigoriadis AE, Wang ZQ, Cecchini MG, Hofstetter W, Felix R, myeloma cells in the bone marrow microenvironment. Cancer Res Fleisch HA et al. c-Fos: a key regulator of osteoclast-macrophage 2006; 66: 6675–6682. lineage determination and bone remodeling. Science 1994; 266: 35 Moreaux J, Legouffe E, Jourdan E, Quittet P, Reme T, Lugagne C 443–448. et al. BAFF and APRIL protect myeloma cells from apoptosis 24 Wang ZQ, Ovitt C, Grigoriadis AE, Mohle-Steinlein U, Ruther U, induced by interleukin 6 deprivation and dexamethasone. Blood Wagner EF. Bone and haematopoietic defects in mice lacking 2004; 103: 3148–3157. c-fos. Nature 1992; 360: 741–745. 36 Moreaux J, Cremer FW, Reme T, Raab M, Mahtouk K, Kaukel P 25 Takayanagi H, Kim S, Koga T, Nishina H, Isshiki M, Yoshida H et al. The level of TACI gene expression in myeloma et al. Induction and activation of the transcription factor NFATc1 cells is associated with a signature of microenvironment (NFAT2) integrate RANKL signaling in terminal differentiation of dependence versus a plasmablastic signature. Blood 2005; 106: osteoclasts. Dev Cell 2002; 3: 889–901. 1021–1030. 26 Oyajobi BO, Franchin G, Williams PJ, Pulkrabek D, Gupta A, 37 Abe M, Kido S, Hiasa M, Nakano A, Oda A, Amou H et al. Munoz S et al. Dual effects of macrophage inflammatory protein- BAFF and APRIL as osteoclast-derived survival factors for 1alpha on osteolysis and tumor burden in the murine 5TGM1 myeloma cells: a rationale for TACI-Fc treatment in patients with model of myeloma bone disease. Blood 2003; 102: 311–319. multiple myeloma. Leukemia 2006; 20: 1313–1315. 27 Han JH, Choi SJ, Kurihara N, Koide M, Oba Y, Roodman GD. 38 Yaccoby S, Pennisi A, Li X, Dillon SR, Zhan F, Barlogie B et al. Macrophage inflammatory protein-1alpha is an osteoclastogenic Atacicept (TACI-Ig) inhibits growth of TACI(high) primary myelo- factor in myeloma that is independent of receptor activator of ma cells in SCID-hu mice and in coculture with osteoclasts. nuclear factor kappaB ligand. Blood 2001; 97: 3349–3353. Leukemia 2008; 22: 406–413. 28 Choi SJ, Cruz JC, Craig F, Chung H, Devlin RD, Roodman GD 39 Sezer O, Heider U, Jakob C, Zavrski I, Eucker J, Possinger K et al. Immunocytochemistry reveals RANKL expression of myeloma et al. Macrophage inflammatory protein 1-alpha is a potential cells. Blood 2002; 99: 4646–4647. osteoclast stimulatory factor in multiple myeloma. Blood 2000; 96: 40 Farrugia AN, Atkins GJ, To LB, Pan B, Horvath N, Kostakis P et al. 671–675. Receptor activator of nuclear factor-kappaB ligand expression by 29 Abe M, Hiura K, Wilde J, Moriyama K, Hashimoto T, Ozaki S et al. human myeloma cells mediates osteoclast formation in vitro and Role for macrophage inflammatory protein (MIP)-1alpha and MIP- correlates with bone destruction in vivo. Cancer Res 2003; 63: 1beta in the development of osteolytic lesions in multiple 5438–5445. myeloma. Blood 2002; 100: 2195–2202. 41 Sezer O, Heider U, Zavrski I, Kuhne CA, Hofbauer LC. RANK 30 Lentzsch S, Gries M, Janz M, Bargou R, Dorken B, Mapara MY. ligand and osteoprotegerin in myeloma bone disease. Blood 2003; Macrophage inflammatory protein 1-alpha (MIP-1 alpha) triggers 101: 2094–2098. migration and signaling cascades mediating survival and 42 Vanderkerken K, De Leenheer E, Shipman C, Asosingh K, Willems proliferation in multiple myeloma (MM) cells. Blood 2003; 101: A, Van Camp B et al. Recombinant osteoprotegerin decreases 3568–3573. tumor burden and increases survival in a murine model of multiple 31 Moore PA, Belvedere O, Orr A, Pieri K, La Fleur DW, Feng P et al. myeloma. Cancer Res 2003; 63: 287–289. BLyS: member of the tumor necrosis factor family and B 43 Terpos E, Szydlo R, Apperley JF, Hatjiharissi E, Politou M, lymphocyte stimulator. Science 1999; 285: 260–263. Meletis J et al. Soluble receptor activator of nuclear factor 32 Hahne M, Kataoka T, Schroter M, Hofmann K, Irmler M, Bodmer JL kappaB ligand-osteoprotegerin ratio predicts survival in multiple et al. APRIL, a new ligand of the tumor necrosis factor family, myeloma: proposal for a novel prognostic index. Blood 2003; 102: stimulates tumor cell growth. J Exp Med 1998; 188: 1185–1190. 1064–1069. 33 Moreaux J, Cremer FW, Reme T, Raab M, Mahtouk K, Kaukel P 44 Terpos E, Mihou D, Szydlo R, Tsimirika K, Karkantaris C, Politou M et al. The level of TACI gene expression in myeloma cells is et al. The combination of intermediate doses of thalidomide with associated with a signature of microenvironment dependence dexamethasone is an effective treatment for patients with versus a plasmablastic signature. Blood 2005; 106: 1021–1030. refractory/relapsed multiple myeloma and normalizes abnormal 34 Tai YT, Li XF, Breitkreutz I, Song W, Neri P, Catley L et al. Role of bone remodeling, through the reduction of sRANKL/osteoprote- B-cell-activating factor in adhesion and growth of human multiple gerin ratio. Leukemia 2005; 19: 1969–1976.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

Leukemia